Light-initiated hydroxylation of lauric acid using hybrid P450 BM3 enzymes.
Ontology highlight
ABSTRACT:
SUBMITTER: Tran NH
PROVIDER: S-EPMC3213755 | biostudies-literature | 2011 Nov
REPOSITORIES: biostudies-literature
Publications
Light-initiated hydroxylation of lauric acid using hybrid P450 BM3 enzymes.
Tran Ngoc-Han NH Huynh Ngoc N Bui Thuba T Nguyen Yen Y Huynh Phuong P Cooper Mary E ME Cheruzel Lionel E LE
Chemical communications (Cambridge, England) 20111005 43
We have developed hybrid P450 BM3 enzymes consisting of a Ru(II)-diimine photosensitizer covalently attached to non-native single cysteine residues of P450 BM3 heme domain mutants. These enzymes are capable, upon light activation, of selectively hydroxylating lauric acid with 40 times higher total turnover numbers compared to the peroxide shunt. ...[more]
Similar Datasets
Project description:We have developed a series of hybrid P450 BM3 enzymes to perform the light-activated hydroxylation of lauric acid. These enzymes contain a Ru(II)-diimine photosensitizer covalently attached to single cysteine residues of mutant P450 BM3 heme domains. The library of hybrid enzymes includes four non-native single cysteine mutants (K97C, Q397C, Q109C and L407C). In addition, mutations around the heme active site, F87A and I401P, were inserted in the Q397C mutant. Two heteroleptic Ru(II) complexes, Ru(bpy)(2)phenA (1) and Ru(phen)(2)phenA (2) (bpy=bipyridine, phen=1,10-phenanthroline, and phenA=5-acetamido-1,10-phenanthroline), are used as photosensitizers. Upon visible light irradiation, the hybrid enzymes display various total turnover numbers in the hydroxylation of lauric acid, up to 140 for the L407C-1 mutant, a 16-fold increase compared to the F87A/Q397C-1 mutant. CO binding studies confirm the ability of the photogenerated Ru(I) compound to reduce the fraction of ferric high spin species present in the mutants upon substrate binding.
| S-EPMC3486690 | biostudies-literature
Project description:Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ?-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ?-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2-2) for 12-hydroxylation with 12-(2)H-substituted lauric acid. However, considerable "metabolic switching" to 11-hydroxylation was observed with [12-(2)H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc. 108, 7074-7078] and the use of tritium KIE analysis with [12-(3)H]lauric acid [Northrop, D. B. (1987) Methods Enzymol. 87, 607-625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ?-hydroxylation ?2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C-H bond-breaking limit the rate of P450 4A11 ?-oxidation.
| S-EPMC4188250 | biostudies-literature
Project description:P450s are heme thiolate enzymes that catalyze the regio- and stereoselective functionalization of unactivated C-H bonds using molecular dioxygen and two electrons delivered by the reductase. We have developed hybrid P450 BM3 heme domains containing a covalently attached Ru(II) photosensitizer in order to circumvent the dependency on the reductase and perform P450 reactions upon visible light irradiation. A highly active hybrid enzyme with improved stability and a modified Ru(II) photosensitizer is able to catalyze the light-driven hydroxylation of lauric acid with total turnover numbers of 935 and initial reaction rate of 125 mol product/(mol enzyme/min).
| S-EPMC3938315 | biostudies-literature
Project description:BACKGROUND:In order to perform selective CH functionalization upon visible light irradiation, Ru(II)-diimine functionalized P450 heme enzymes have been developed. The sL407C-1 enzyme containing the Ru(bpy)2PhenA (bpy=2,2'-bipyridine and PhenA=5-acetamido-1,10-phenanthroline) photosensitizer (1) covalently attached to the non-native single cysteine L407C of the P450BM3 heme domain mutant, displays high photocatalytic activity in the selective CH bond hydroxylation of several substrates. METHODS:A combination of X-ray crystallography, site-directed mutagenesis, transient absorption measurements and enzymatic assays was used to gain insights into its photocatalytic activity and electron transfer pathway. RESULTS:The crystal structure of the sL407C-1 enzyme was solved in the open and closed conformations revealing a through-space electron transfer pathway involving highly conserved, F393 and Q403, residues. Several mutations of these residues (F393A, F393W or Q403W) were introduced to probe their roles in the overall reaction. Transient absorption measurements confirm rapid electron transfer as heme reduction is observed in all four hybrid enzymes. Compared to the parent sL407C-1, photocatalytic activity was negligible in the dF393A-1 enzyme while 60% increase in activity with total turnover numbers of 420 and 90% product conversion was observed with the dQ403W-1 mutant. CONCLUSIONS:In the sL407C-1 enzyme, the photosensitizer is ideally located to rapidly deliver electrons, using the naturally occurring electron transfer pathway, to the heme center in order to activate molecular dioxygen and sustain photocatalytic activity. GENERAL SIGNIFICANCE:The results shed light on the design of efficient light-driven biocatalysts and the approach can be generalized to other members of the P450 superfamily.
| S-EPMC5071167 | biostudies-literature
Project description:The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88-99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.
| S-EPMC7261786 | biostudies-literature
Project description:While the mechanism of the P450-catalyzed oxidative hydroxylation of organic compounds has been studied in detail for many years, less is known about sulfoxidation. Depending upon the structure of the respective substrate, heme-Fe?O (Cpd I), heme-Fe(III)-OOH (Cpd 0), and heme-Fe(III)-H2O2 (protonated Cpd 0) have been proposed as reactive intermediates. In the present study, we consider the transformation of isosteric substrates via sulfoxidation and oxidative hydroxylation, respectively, catalyzed by regio- and enantioselective mutants of P450-BM3 which were constructed by directed evolution. 1-Thiochromanone and 1-tetralone were used as the isosteric substrates because, unlike previous studies involving fully flexible compounds such as thia-fatty acids and fatty acids, respectively, these compounds are rigid and cannot occur in a multitude of different conformations and binding modes in the large P450-BM3 binding pocket. The experimental results comprising activity and regio- and enantioselectivity, flanked by molecular dynamics computations within a time scale of 300 ns and QM/MM calculations of transition-state energies, unequivocally show that heme-Fe?O (Cpd I) is the common catalytically active intermediate in both sulfoxidation and oxidative hydroxylation.
| S-EPMC7307895 | biostudies-literature
Project description:In this study, we identified two P450 enzymes (CYP5150AP3 and CYP5150AN1) from Thanatephorus cucumeris NBRC 6298 by combination of transcriptome sequencing and heterologous expression in Pichia pastoris The biotransformation of 11-deoxycortisol and testosterone by Pichia pastoris whole cells coexpressing the cyp5150ap3 and por genes demonstrated that the CYP5150AP3 enzyme possessed steroidal 7β-hydroxylase activities toward these substrates, and the regioselectivity was dependent on the structures of steroidal compounds. CYP5150AN1 catalyzed the 2β-hydroxylation of 11-deoxycortisol. It is interesting that they display different regioselectivity of hydroxylation from that of their isoenzyme, CYP5150AP2, which possesses 19- and 11β-hydroxylase activities.IMPORTANCE The steroidal hydroxylases CYP5150AP3 and CYP5150AN1 together with the previously characterized CYP5150AP2 belong to the CYP5150A family of P450 enzymes with high amino acid sequence identity, but they showed completely different regioselectivities toward 11-deoxycortisol, suggesting the regioselectivity diversity of steroidal hydroxylases of CYP5150 family. They are also distinct from the known bacterial and fungal steroidal hydroxylases in substrate specificity and regioselectivity. Biocatalytic hydroxylation is one of the important transformations for the functionalization of steroid nucleus rings but remains a very challenging task in organic synthesis. These hydroxylases are useful additions to the toolbox of hydroxylase enzymes for the functionalization of steroids at various positions.
| S-EPMC6715852 | biostudies-literature
Project description:Ru(II)-diimine complexes covalently attached near the heme active site of P450 BM3 enzymes have been used to rapidly inject electrons and drive selective C-H functionalization upon visible light irradiation. Herein, we have generated a series of hybrid P450 BM3 enzymes containing a photosensitizer of general formula [Ru(4,4'-X2bpy)2(PhenA)]2+ where X = Cl, H, tBu, Me OPhe, OMe, or NMe2, bpy = 2,2'-bipyridine, and PhenA = 5-acetamido-1,10-phenanthroline. We then probed the effect of electron-withdrawing and -donating groups at the para position of the 4,4'-X2bpy ligands on the corresponding hybrid enzymes photocatalytic activity. A 3-fold improvement in initial reaction rate was noted when varying the substituent from Cl to tBu, however, the reaction rates decrease thereafter with the more electron donating groups. In order to rationalize those effects, we investigated the variation of the substituent on the photophysical properties of the corresponding [Ru(4,4'-X2bpy)2(bpy)]2+ model complexes. Several linear correlations were established between the E(III/II) potential, the MLCT emission, and absorption energies as well as the logarithm of the luminescence quenching rate vs the summative Brown-Okamoto parameter (??p+). Moreover, a downward curved Hammett plot is observed with the hybrid enzyme initial reaction rate revealing mechanistic details about the overall light-driven enzymatic process.
| S-EPMC5535733 | biostudies-literature
Project description:High-valent iron-oxo species are thought to be intermediates in the catalytic cycles of oxygenases and peroxidases. An attractive route to these iron-oxo intermediates involves laser flash-quench oxidation of ferric hemes, as demonstrated by our work on the ferryl (compound II) and ferryl porphyrin radical cation (compound I) intermediates of horseradish peroxidase. Extension of this work to include cytochrome P450-BM3 (CYP102A1) has required covalent attachment of a Ru(II) photosensitizer to a nonnative cysteine near the heme (RuIIK97C-FeIIIP450), in order to promote electron transfer from the Fe(III) porphyrin to photogenerated Ru(III). The conjugate was structurally characterized by X-ray crystallography (2.4 ? resolution; Ru-Fe distance, 24 ?). Flash-quench oxidation of the ferric-aquo heme produces an Fe(IV)-hydroxide species (compound II) within 2 ms. Difference spectra for three singly oxidized P450-BM3 intermediates were obtained from kinetics modeling of the transient absorption data in combination with generalized singular value decomposition analysis and multiexponential fitting.
| S-EPMC2973866 | biostudies-literature
Project description:Physiological plasticity in a polluted system: A case of an uncultured bacterium and its cypome
| PRJEB38781 | ENA